Molecular design of inhibitors of in vitro oriC DNA replication based on the potential to block the ATP binding of DnaA protein

Tohru Mizushima, Shigeki Sasaki, Hiroko Ohishi, Masakatsu Kobayashi, Tsutomu Katayama, Takeyoshi Miki, Minoru Maeda, Kazuhisa Sekimizu

Research output: Contribution to journalArticle

36 Citations (Scopus)

Abstract

DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by binding to ATP. We earlier reported that 3- acetoxy-2,2'-bi-1H-indol inhibited the ATP binding to DnaA protein (Sasaki, S., Mizushima, T., Hashimoto, T., Maeda, M., and Sekimizu, K. (1994) Bioorg. Med. Chem. Lett. 4, 1771-1774). In the present study, derivatives of 3- acetoxy-2,2'-bi-1H-indol with different lengths of aliphatic chains at the 3- O position were synthesized, and their potential to inhibit the ATP binding to DnaA protein was examined. Elongation of the aliphatic chain resulted in inhibition of the ATP binding to DnaA protein at lower concentrations. Among the derivatives, 3-[N-(11-carboxyundecyl)] carbamoylmethoxy-2,2'-bi-1H-indol (structure 7 (3-CUCM-BI)) exhibited the most potent inhibition with an IC50 value of 7 μM. The mode of the inhibition was competitive. We further demonstrated that structure 7 (3-CUCM-BI) inhibited DNA replication of the oriC plasmid in a system reconstituted from purified proteins. This inhibition was specific for the initiation of DNA replication rather than for the elongation. The inhibition was overcome by preincubation of DnaA protein with ATP. Furthermore, structure 7 (3-CUCM-BI) showed little inhibition on DNA synthesis in the ABC primosome system. We propose that structure 7 (3- CUCM-BI) functions in the in vitro oriC DNA replication by inhibiting the ATP binding to DnaA protein.

Original languageEnglish
Pages (from-to)25178-25183
Number of pages6
JournalJournal of Biological Chemistry
Volume271
Issue number41
DOIs
Publication statusPublished - 1996
Externally publishedYes

Fingerprint

DNA Replication
Carrier Proteins
Adenosine Triphosphate
DNA
Proteins
Elongation
Derivatives
Peptide Initiation Factors
In Vitro Techniques
Escherichia coli
Inhibitory Concentration 50
Plasmids

ASJC Scopus subject areas

  • Biochemistry

Cite this

Molecular design of inhibitors of in vitro oriC DNA replication based on the potential to block the ATP binding of DnaA protein. / Mizushima, Tohru; Sasaki, Shigeki; Ohishi, Hiroko; Kobayashi, Masakatsu; Katayama, Tsutomu; Miki, Takeyoshi; Maeda, Minoru; Sekimizu, Kazuhisa.

In: Journal of Biological Chemistry, Vol. 271, No. 41, 1996, p. 25178-25183.

Research output: Contribution to journalArticle

Mizushima, T, Sasaki, S, Ohishi, H, Kobayashi, M, Katayama, T, Miki, T, Maeda, M & Sekimizu, K 1996, 'Molecular design of inhibitors of in vitro oriC DNA replication based on the potential to block the ATP binding of DnaA protein', Journal of Biological Chemistry, vol. 271, no. 41, pp. 25178-25183. https://doi.org/10.1074/jbc.271.41.25178
Mizushima, Tohru ; Sasaki, Shigeki ; Ohishi, Hiroko ; Kobayashi, Masakatsu ; Katayama, Tsutomu ; Miki, Takeyoshi ; Maeda, Minoru ; Sekimizu, Kazuhisa. / Molecular design of inhibitors of in vitro oriC DNA replication based on the potential to block the ATP binding of DnaA protein. In: Journal of Biological Chemistry. 1996 ; Vol. 271, No. 41. pp. 25178-25183.
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T1 - Molecular design of inhibitors of in vitro oriC DNA replication based on the potential to block the ATP binding of DnaA protein

AU - Mizushima, Tohru

AU - Sasaki, Shigeki

AU - Ohishi, Hiroko

AU - Kobayashi, Masakatsu

AU - Katayama, Tsutomu

AU - Miki, Takeyoshi

AU - Maeda, Minoru

AU - Sekimizu, Kazuhisa

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AB - DnaA protein, the initiation factor for chromosomal DNA replication in Escherichia coli, is activated by binding to ATP. We earlier reported that 3- acetoxy-2,2'-bi-1H-indol inhibited the ATP binding to DnaA protein (Sasaki, S., Mizushima, T., Hashimoto, T., Maeda, M., and Sekimizu, K. (1994) Bioorg. Med. Chem. Lett. 4, 1771-1774). In the present study, derivatives of 3- acetoxy-2,2'-bi-1H-indol with different lengths of aliphatic chains at the 3- O position were synthesized, and their potential to inhibit the ATP binding to DnaA protein was examined. Elongation of the aliphatic chain resulted in inhibition of the ATP binding to DnaA protein at lower concentrations. Among the derivatives, 3-[N-(11-carboxyundecyl)] carbamoylmethoxy-2,2'-bi-1H-indol (structure 7 (3-CUCM-BI)) exhibited the most potent inhibition with an IC50 value of 7 μM. The mode of the inhibition was competitive. We further demonstrated that structure 7 (3-CUCM-BI) inhibited DNA replication of the oriC plasmid in a system reconstituted from purified proteins. This inhibition was specific for the initiation of DNA replication rather than for the elongation. The inhibition was overcome by preincubation of DnaA protein with ATP. Furthermore, structure 7 (3-CUCM-BI) showed little inhibition on DNA synthesis in the ABC primosome system. We propose that structure 7 (3- CUCM-BI) functions in the in vitro oriC DNA replication by inhibiting the ATP binding to DnaA protein.

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